5-CNAC as oral delivery agent for parathyroid hormone fragments

ABSTRACT

Pharmaceutical compositions for the effective oral delivery of a parathyroid hormone, PTH, as well as methods for administration of the compositions are provided. Additionally, methods for stimulating new bone formation and treating and/or preventing osteoporosis are also provided.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the oral delivery of parathyroidhormone (PTH). The mammalian parathyroid hormones, e.g. human (hPTH),bovine (bPTH) and porcine (pPTH), are single polypeptide chains of 84amino acid residues having molecular weights of approximately 9500.Specifically, the present invention relates to PTH fragmentsincorporating at least the first 28 N-terminal amino acid residues (PTH(1-28)) up to and including the first 41 N-terminal amino acid residues(PTH (1-41)). More particularly, the invention is directed topharmaceutical compositions for the oral delivery of PTH, saidcompositions comprising PTH (1-28) to (1-41) andN-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC).

2. Description of the Related Art

PTH studies done in animals and humans with PTH, PTH-related peptides,and PTH analogues have demonstrated its usefulness in increasing boneformation and bone resorption and have prompted interest in its use forthe treatment of osteoporosis and related bone disorders. However, theoral delivery of PTH in mammals has proven difficult due, at least inpart, to the insufficient stability of PTH in the gastrointestinal tractas well as the inability of PTH to be readily transported through theintestinal walls into the blood stream.

U.S. Pat. No. 5,773,647 (the '647 patent) describes 193 carriercompounds useful for the delivery of active agents, including PTH. Oneof the carrier compounds expressly described therein isN-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC) having the formula

Example 2 in Column 6 of the '647 patent describes the preparation of 11different dosing compositions some intracolonic (IC) and some oralgavage (PO) each containing parathyroid hormone and a carrier, thecarrier being different for each composition. An IC dosing compositionwas prepared using 5-CNAC as the carrier. Example 3 therein describes invivo tests carried out dosing male Sprague-Dawley rats with the dosingsolutions prepared in Example 2. Blood samples were collected and theserum PTH concentration was quantified for each rat.

Surprisingly, it has now been found that 5-CNAC in combination withspecific PTH fragments, i.e. PTH fragments incorporating at least thefirst 28 N-terminal aminoacid residues (PTH (1-28)) up to and includingthe first 41 N-terminal amino acid residues (PTH (1-41)) when orallyadministered gives unexpectedly high PTH serum levels relative to otherPTHs and other carriers and provide a sharp C_(max) allowing for a boneformation effect.

SUMMARY OF THE INVENTION

Accordingly, the present invention is directed to pharmaceuticalcompositions suitable for oral delivery of PTH fragments and to methodsof administering such compositions.

Specifically, the instant invention is directed to a pharmaceuticalcomposition for oral delivery comprising a therapeutically effectiveamount of a PTH fragment and 5-CNAC, said PTH fragment selected from PTH(1-28) to PTH (1-41). Preferably, the PTH is human parathyroid hormone,hPTH.

In another embodiment, the invention is directed to a method for orallyadministering an effective dose of PTH comprising orally administeringto a patient in need of PTH a pharmaceutical composition comprising atherapeutically effective amount of a PTH fragment and 5-CNAC, said PTHfragment selected from PTH (1-28) to PTH (1-41).

The invention is also directed to a method of stimulating new boneformation comprising orally administering to a patient in need of newbone formation a pharmaceutical composition comprising a therapeuticallyeffective amount of a PTH fragment and 5-CNAC, said PTH fragmentselected from PTH (1-28) to PTH (1-41).

In a further embodiment, the invention is directed to a method oftreatment or prevention of osteoporosis comprising orally administeringto a patient in need of said treatment or prevention a pharmaceuticalcomposition comprising a therapeutically effective amount of a PTHfragment and 5-CNAC, said PTH fragment selected from PTH (1-28) to PTH(1-41).

In a still further embodiment, the invention is directed to the use of5-CNAC for the preparation of a pharmaceutical composition suitable forthe oral delivery of PTH fragments selected from PTH (1-28) to PTH(1-41).

Further features and advantages of the invention will become apparentfrom the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The PTH fragments can be of any parathyroid hormone, particularlymammalian parathyroid hormone, e.g. human (hPTH), bovine (bPTH), andporcine (pPTH) and particularly hPTH and will incorporate at least thefirst 28 N-terminal residues (PTH (1-28)) up to and including the first41 N-terminal residues (PTH (1-41)) and include without limitation PTH(1-28), PTH (1-31) PTH (1-34), PTH (1-37), PTH (1-38) and PTH (141).Human parathyroid hormone (1-34) is particularly preferred. Theseparathyroid hormone fragments are commercially available or can beobtained recombinantly or by peptide synthesis.

For purposes of the instant invention, the 5-CNAC, i.e.N-(5-chlorosalicyloyl)-8-aminocaprylic acid, can be the free acid,analogs thereof, its monosodium and disodium salts, ethanol solvates ofthe sodium salts and the monohydrates of the sodium salts and anycombinations thereof. The free acid, the disodium salt of 5-CNAC and themonohydrate thereof are particularly useful.N-(5-chlorosalicyloyl)-8-aminocaprylic acid is described in theaforementioned '647 patent, the contents of which are herebyincorporated by reference, and can be made by methods described therein.The sodium salts and alcohol solvates and hydrates thereof are describedin WO 00/059863, along with methods for preparing them.

The disodium salt may be prepared from the ethanol solvate byevaporating or drying the ethanol solvate by methods known in the art toform the anhydrous disodium salt. Drying is generally carried out at atemperature of from about 80 to about 120° C., preferably from about 85to about 90° C., and most preferably at about 85° C. The drying step isgenerally performed at a pressure of 26″ Hg or greater. The anhydrousdisodium salt generally contains less than about 5% by weight of ethanoland preferably less than about 2% by weight of ethanol, based on 100%total weight of anhydrous disodium salt.

The disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid canalso be prepared by making a slurry ofN-(5-chlorosalicyloyl)-8-aminocaprylic acid in water and adding twomolar equivalents of aqueous sodium hydroxide, sodium alkoxide or thelike. Suitable sodium alkoxides include, but are not limited to, sodiummethoxide, sodium ethoxide, and combinations thereof.

A still further method of preparing the disodium salt is by reactingN-(5-chlorosalicyloyl)-8-aminocaprylic acid with one molar equivalent ofsodium hydroxide to form a monosodium salt and then adding an additionalone molar equivalent of sodium hydroxide to yield the disodium salt.

The disodium salt can be isolated as a solid by concentrating thesolution containing the disodium salt to a thick paste by vacuumdistillation. This paste may be dried in a vacuum oven to obtain thedisodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid as a solid.The solid can also be isolated by spray drying an aqueous solution ofthe disodium salt.

The ethanol solvates, as described in the aforementioned WO 00/059863,include, but are not limited to, a molecular or ionic complex ofmolecules or ions of ethanol solvent with molecules or ions of thedisodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid. Typically,the ethanol solvate contains about one ethanol molecule or ion for everymolecule of disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylicacid.

The ethanol solvate of the disodium salt ofN-(5-chlorosalicyloyl)-8-aminocaprylic acid can be prepared bydissolving N-(5-chlorosalicyloyl)8-aminocaprylic acid in ethanol.Typically, each gram of N-(5-chlorosalicyloyl)-8-aminocaprylic acid isdissolved in from about 1 to about 50 mL of ethanol and generally, fromabout 2 to about 10 mL of ethanol. TheN-5-chlorosalicyloyl)-8-aminocaprylic acid/ethanol solution is thenreacted with a molar excess of a sodium containing salt, such as amonosodium containing salt, relative toN-(5-chlorosalicyloyl)8-aminocaprylic acid, i.e. for every mole ofN-(5-chlorosalicyloyl)-8-aminocaprylic acid there is more than one moleof sodium cations, yielding the ethanol solvate. Suitable monosodiumsalts include, but are not limited to, sodium hydroxide; sodiumalkoxides, such as sodium methoxide and sodium ethoxide; and anycombination of the foregoing. Preferably, at least about two molarequivalents of the monosodium containing salt are added to the ethanolsolution, i.e. for every mole of N-(5-chlorosalicyloyl)-8-aminocaprylicacid there is at least about two moles of sodium cations. Generally, thereaction is performed at or below the reflux temperature of the mixture,such as at ambient temperature. The ethanol solvate is then recovered bymethods known is the art, such as, concentration of the resulting slurryat atmospheric distillation, cooling the concentrated slurry andfiltering the solid. The recovered solid can then be vacuum dried toobtain the ethanol solvate.

The hydrates of the disodium salts of theN-(5-chlorosalicyloyl)-8-aminocaprylic acid may be prepared by dryingthe ethanol solvate to form an anhydrous disodium salt, as describedabove, and hydrating the anhydrous disodium salt. Preferably, themonohydrate of the disodium salt is formed. Since the anhydrous disodiumsalt is very hydroscopic, the hydrate forms upon exposure to atmosphericmoisture. Generally, the hydrating step is performed at from aboutambient temperature to about 50° C., preferably ambient temperature toabout 30° C. and in an environment having at least 50% relativehumidity. Alternatively, the anhydrous disodium salt may be hydratedwith steam.

The amount of PTH fragment to be administered is generally an amounteffective to stimulate new bone formation i.e. a therapeuticallyeffective amount. This amount will necessarily vary with the age, size,sex and condition of the subject to be treated, the nature and severityof the disorder to be treated and the like. However, the amount can beless than that amount when a plurality of the compositions are to beadministered, i.e., the total effective amount can be administered incumulative dosage units. The amount of PTH can also be more than theeffective amount when the composition provides sustained release of thepharmacologically active agent. The total amount of PTH to be used canbe determined by methods known to those skilled in the art. However, ingeneral, satisfactory results will be obtained systemically at dailydosages of from about 0.001 μg/kg to about 10 mg/kg animal body weight,preferably 1 μg/kg to about 6 μg/kg body weight.

The pharmaceutical compositions of the present invention typicallycontain a delivery effective amount of 5-CNAC, i.e. an amount sufficientto deliver the PTH for the desired effect Generally, the 5-CNAC ispresent in an amount of 2.5% to 99.4% by weight, more preferably 25% to50% by weight of the total composition.

Oral administration of the pharmaceutical compositions according to theinvention can be accomplished regularly, e.g. once or more on a daily orweekly basis; intermittently, e.g. irregularly during a day or week; orcyclically, e.g. regularly for a period of days or weeks followed by aperiod without administration.

The dosage form of the pharmaceutical compositions of the instantinvention can be any known form, e.g. liquid or solid dosage forms.

The liquid dosage forms include solution emulsions, suspensions, syrupsand elixirs. In addition to the PTH and 5-CNAC, the liquid formulationsmay also include inert excipients commonly used in the art such as,solubilizing agents e.g. ethanol; oils such as cottonseed, castor andsesame oils; wetting agents; emulsifying agents; suspending agents;sweeteners; flavorings; and solvents such as water.

The solid dosage forms include capsules, soft-gel capsules, tablets,caplets, powders, granules or other solid oral dosage forms, all ofwhich can be prepared by methods well known in the art.

The pharmaceutical compositions may additionally comprise additives inamounts customarily employed including, but not limited to, a pHadjuster, a preservative, a flavorant, a taste-masking agent, afragrance, a humectant, a tonicifier, a colorant, a surfactant, aplasticizer, a lubricant such as magnesium stearate, a flow aid, acompression aid, a solubilizer, an excipient, a diluent such asmicrocrystalline cellulose, e.g. Avicel PH 102 supplied by FMCcorporation, or any combination thereof. Other additives may includephosphate buffer salts, citric acid, glycols, and other dispersingagents.

The composition may also include one or more enzyme inhibitors, such asactinonin or epiactinonin and derivatives thereof; aprotinin, Trasyloland Bowman-Birk inhibitor.

Further, a transport inhibitor, i.e. a ρ-glycoprotein such asKetoprofin, may be present in the compositions of the present invention.

The solid pharmaceutical compositions of the instant invention can beprepared by conventional methods e.g. by blending a mixture of the PTHfragment, the 5-CNAC, and any other ingredients, kneading, and fillinginto capsules or, instead of filling into capsules, molding followed byfurther tableting or compression-molding to give tablets. In addition, asolid dispersion may be formed by known methods followed by furtherprocessing to form a tablet or capsule.

Preferably, the ingredients in the pharmaceutical compositions of theinstant invention are homogeneously or uniformly mixed throughout thesolid dosage form.

Parathyroid hormones are indicated for preventing or treating all boneconditions which are associated with increased calcium depletion orresorption or in which stimulation of bone formation and calciumfixation in the bone is desirable, e.g. osteoporosis of various genesis(e.g. juvenile, menopausal, post-menopausal, post-traumatic, caused byold age or by corticoid-steroid therapy a or inactivity), fractures,osteopathy, including acute and chronic states associated with skeletaldemineralization, osteo-malacia, periodontal bone loss or bone loss dueto arthritis or osteoarthritis or cancer (e.g. bone; metastis) or fortreating hypoparathyroidism.

Parathyroid hormones are particularly indicated for preventing ortreating osteoporosis of various genesis.

According to a further embodiment of the invention, the PTH may, beemployed as adjunct or adjuvant to other therapy, e.g. a therapy using abone resorption inhibitor, for example as in osteoporosis therapy, inparticular a therapy employing calcium, a calcitonin or an analogue orderivative thereof, e.g. salmon, eel or human calcitonin, a steroidhormone, e.g. an estrogen, a partial estrogen agonist orestrogen-gestagen combination, a SERM (Selective Estrogen ReceptorModulator) e.g. raloxifene, lasofoxifene, TSE-424, FC1271, Tibolone(Livial®), vitamin D or an analogue thereof or an activator of PTHrelease, or bisphosphonates, e.g. clodronic acid, etidronic acid,pamidronic acid, aledronic acid, ibandronic acid, zoledronic acid,risedronic acid or tiludronic acid and salts and hydrates thereof.

When the PTH is administered in conjunction with, e.g. as an adjuvant tobone resorption inhibition therapy, dosages for the co-administeredinhibitor will of course vary depending on the type of inhibitor drugemployed, e.g. whether it is a steroid or a calcitonin, on the conditionto be treated, whether it is a curative or preventive therapy, on theregimen and so forth.

The oral administration of the present invention may be to any animal inneed thereof, including, but not limited to, mammals, such as rodents,cows, pigs, dogs, cats, and primates, particularly humans.

The following examples serve to further illustrate the invention.

EXAMPLE 1

The following capsules are prepared as follows:

Capsules prepared from 800 μg hPTH* (Capsule 1A)

Capsules prepared from 400 mg 5-CNAC**/800 μg hPTH* (Capsule 1B)

*The PTH fragment is human parathyroid hormone, fragment 1-34commercially available from Sigma. **The 5-CNAC is the disodium salt ofN-(5-chlorosalicyloyl)8-aminocaprylic acid.

The hPTH only capsules are prepared by weighing out 400 μg of the hPTHand placing it directly into each capsule. The hPTH/5-CNAC capsules areprepared as dry blends by weighing out the individual componentsblending them together to make a homogeneous mix and then hand filling400 mg of the mix into each capsule.

EXAMPLE 2 Primate Administration

The capsules prepared in Example 1 are administered to Rhesus monkeys asfollows: four monkeys in a group are each dosed with one capsuleprepared as in Example 1 as follows:

The Rhesus monkeys fast overnight prior to dosing and are restrained inchairs fully conscious, for the duration of the study period. Thecapsules are orally administered via a gavage tube followed by 10 mL ofwater.

Blood samples are collected at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5,and 6 hours after administration. Plasma hPTH levels are determined byradioimmunoassay. The primate plasma PTH results from each group ofmonkeys are averaged and the maximum mean plasma calculated. The resultsfor the PTH only group are reported in Table 1 and the results for thehPTH/5-CNAC group are reported in Table 2. TABLE 1 hPTH ONLY hPTH PLASMACONCENTRATIONS (pg/mL) AFTER ORAL ADMINISTRATION TO THE RHESUS MONKEYDose: 1 Capsule 1D Animal Time [hours] no. 0 0.25 0.50 0.75 1 1.5 2 3 45 6 R927 0 0 0 0 0 0 0 0 0 0 0 S982 0 0 0 0 0 0 0 0 0 0 0 Mean 0 0 0 0 00 0 0 0 0 0 SD 0 0 0 0 0 0 0 0 0 0 0 SEM 0 0 0 0 0 0 0 0 0 0 0LLOQ = 25 pg/mL, concentrations below LLOQ were set to zero.

TABLE 2 hPTH/5-CNAC hPTH PLASMA CONCENTRATIONS (pg/mL) AFTER ORALADMINISTRATION TO THE RHESUS MONKEY Dose: 1 Capsule 1B Ani- mal Time[hours] no. 0 0.25 0.50 0.75 1 1.5 2 3 4 5 6 R944 0 83 191 300 360 262154 35 0 0 0 S963 0 127 332 663 1258 150 34 0 0 0 0 Mean 0 105 262 482809 206 94 17 0 0 0 SD 0 31 100 257 635 79 85 25 0 0 0 SEM 0 22 71 182449 56 60 17 0 0 0

As can be seen from the data in Tables-1 and 2, the 5-CNAC significantlyfacilitates the oral 4 delivery of the hPTH fragment In addition, thedata in Table 2 indicate a sharp C_(max) in the PTH plasma profileallowing for a bone formation effect.

The foregoing embodiments and examples are given merely to illustratethe instant invention and are not intended to be limiting. Numerousother embodiments and variations are within the scope of the inventionand readily accessible to those skilled in the art.

1. A pharmaceutical composition for oral delivery comprising a PTHfragment and N-(5-chlorosalicviovl)-8-aminocaprylic acid andpharmaceutically acceptable salts and esters thereof, said PTH fragmentselected from PTH (1-28) to PTH (1-41).
 2. A pharmaceutical compositionaccording to claim 1 wherein the PTH fragment is selected from PTH(1-28), PTH (1-31), PTH (1-34), PTH (1-37), PTH (1-38) and PTH (1-41).3. A pharmaceutical composition according to claim 1 wherein the PTHfragment is PTH (1-34).
 4. A pharmaceutical composition according toclaim 1 wherein the PTH fragment is recombinant PTH.
 5. A pharmaceuticalcomposition according to claim 3 wherein the PTH fragment is recombinantPTH.
 6. A pharmaceutical composition according to claim 1 wherein thePTH fragment is human parathyroid hormone.
 7. A pharmaceuticalcomposition according to claim 6 wherein the human parathyroid hormoneis hPTH (1-34).
 8. A pharmaceutical composition for oral deliverycomprising a PTH fragment and a compound selected from the free acid,the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid and themonohydrate thereof, said PTH fragment selected from PTH (1-28) to PTH(1-41).
 9. A pharmaceutical composition according to claim 8 wherein thecompound is N-(5-chloro-salicyloyl)-8-aminocaprylic acid.
 10. Apharmaceutical composition for oral delivery comprising a PTH fragmentand the disodium salt of N-(5-chloro-salicyloyl)-8-aminocaprylic acid.11. A method for orally administering an effective dose of PTHcomprising orally administering to a patient in need of PTH apharmaceutical composition comprising a therapeutically effective amountof a PTH fragment and 5-CNAC, said PTH fragment selected from PTH(1-28)-PTH (1-41).
 12. A method according to claim 11 wherein the PTH isselected from PTH (1-28), PTH (1-31), PTH (1-34), PTH (1-37), PTH (1-38)and PTH (1-41).
 13. A method according to claim 11 wherein the PTH ishPTH (1-34).
 14. A method according to claim 11 wherein the PTH isrecombinant PTH.
 15. A method according to claim 11 wherein the PTH isrecombinant hPTH.
 16. A method according to claim 11 wherein the 5-CNACis selected from the free acid, the disodium salt ofN-(5-chlorosalicyloyl)-8-aminocaprylic acid and the monohydrate thereof.17. A method according to claim 11 wherein the 5-CNAC isN-(5-chloro-salicyloyl)-8-aminocaprylic acid.
 18. A method according toclaim 11 wherein the 5-CNAC is the disodium salt ofN-(5-chloro-salicyloyl)-8-aminocaprylic acid.
 19. A method ofstimulating new bone formation comprising orally administering to apatient in need of new bone formation a pharmaceutical compositioncomprising a therapeutically effective amount of a PTH fragment and5-CNAC, said PTH fragment selected from PTH (1-28)-PTH (1-41).
 20. Amethod of treatment or prevention of osteoporosis comprising orallyadministering to a patient in need of said treatment or prevention apharmaceutical composition comprising a therapeutically effective amountof a PTH fragment and 5-CNAC, said PTH fragment selected from PTH(1-28)-PTH (1-41).
 21. The use of 5-CNAC for the preparation of apharmaceutical composition suitable for the oral delivery of PTHfragments selected from PTH (1-28)-PTH (1-41).